1. Field of the Invention
The present invention relates to a method for preparing halo-L-tryptophan from haloindole using a microorganism.
2. Discussion of the Background
Halo-L-tryptophan, for example 5-chloro-L-tryptophan, is useful as a raw material for pharmaceutical products and as a synthetic intermediate for pharmaceutical products.
Methods for producing L-tryptophan from indole using microorganisms include a method using Escherichia coli (French Patent No. 1207437) and a method using a microorganism belonging to the genera Proteus, Erwinia, Pseudomonas or Aerobacter (Japanese Patent Publication No. 46348/1972). Methods for producing 5-hydroxy-L-tryptophan from 5-hydroxyindole include a method using a microorganism belonging to the genera Proteus, Erwinia, Pseudomonas or Aerobacter (Japanese Patent Publication No. 46348/1972) as well as methods using microorganisms such as Bacillus subtilis, Corynebacterium hydrocarboclustus, Arthrobacter paraffinens, Micrococcus ureae, Brevibacterium ketoglutamicum, Hansenula anomala and Candida tropicalis (Japanese Patent Publication No. 46349/1972). Additionally, a method for producing 5-amino-L-tryptophan from 5-aminoindole or for producing 5-methyl-L-tryptophan from 5-methylindole is also known (Japanese Patent Publication No. 9760/1977).
Generally, optically active halo-L-tryptophan can be produced by the combination of a chemical synthesis of N-acetyl-halo-DL-tryptophan and the optical resolution of halo-L-tryptophan by aminoacylase, that is, preparing halo-DL-tryptophan by a chemical synthetic method and subsequently acetylating halo-DL-tryptophan into N-acetylhalo-DL-tryptophan, which is further subjected to an optical resolution method with aminoacylase. However, the method requires complicated processes and the presence of residual N-acetyl-halo-D-tryptophan is disadvantageous.
No method is known for producing halo-L-tryptophan from haloindole. Accordingly, there remains a need for an method for producing halo-L-tryptophan from haloindole on an industrially efficient scale which overcomes the disadvantages of the known methods for preparing halo-L-tryptophan.
The present invention relates to a method for preparing a halo-L-tryptophan using a microorganism.
In particular, the present invention relates to a method for preparing halo-L-tryptophan comprising contacting a haloindole with a microorganism capable of producing halo-L-tryptophan from (a) a mixture comprising haloindole, pyruvic acid and ammonia; or (b) a mixture comprising haloindole and a source of pyruvic acid and ammonia.
In another embodiment, the present invention relates to a method for preparing of halo-L-tryptophan wherein L-tryptophan or a surfactant are also present in a culture medium.
The present inventor has considered that pyridoxal-5xe2x80x2-phosphate is the coenzyme of the enzyme that is involved in the present invention. The inventor have also considered that the molecule of pyridoxal-5xe2x80x2-phosphate might be removed from the enzyme during the cell collection. Therefore, the inventor has added pyridoxal-5xe2x80x2-phosphate into the reaction mixture to supplement the coenzyme. However, in another experiment, the inventor has found that supplementation of pyridoxal-5xe2x80x2-phosphate is not necessary for the present invention.
L-tryptophan is added into the culture medium. The inventor has found that the enzyme is induced by L-trypotphan and added it into the culture medium, not the reaction mixture. Surfactant is also added into the culture medium. The inventor has found that the enzyme converts L-tryptophan into indole (the reverse reaction) and the resulting indole has toxic effects on the cells. The surfactant surrounds the indole to form a micelle, thereby rendering it non-toxic. Therefore, the surfactant is not added into the reaction mixture.
In another embodiment, the present invention relates to a method for preparing a halo-L-tryptophan wherein L-tryptophan or a surfactant are also present in the culture medium.
A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description.
The microorganism employed in the present invention may be any microorganism capable of producing halo-L-tryptophan at a high optical purity from (a) a combination of haloindole, pyruvic acid and ammonia; or (b) a combination of haloindole and a source supplying pyruvic acid and ammonia. The source providing pyruvic acid and ammonia may be L-serine, L-cysteine, O-methyl-L-serine, O-benzyl-L-serine, S-methylcysteine, S-benzylcysteine, for example.
The microbial materials, regardless of origin or purity, may be employed as cells in the free state or as cells immobilized on a support such as by physical adsorption or entrapment. Immobilized cells or immobilized treated cell matter can be used, for example, by using entrapment in carrageenan or polyacrylamide or adsorption onto membranes of polyether sulfone or regenerated cellulose, for example. When intact cells are used in the present invention, the culture per se or intact bacterial cells collected from the culture may be used. The microbial cells may be used in the form of dried cells such as lyophilized, spray-dried or heat-dried cells, or in the form of treated cell matter.
As used herein, the term xe2x80x9ctreated cell matterxe2x80x9d means the biological material, such as cell walls, membranes, nuclei, proteins, etc. which result from the subjection of intact cells to mechanical or chemical disruption. Preferred methods of disrupting intact cells by mechanical means include disruption by ultrasonication, glass beads, pressing and freeze-drying. Pressing in a French Press is particularly preferred. Preferred methods of disrupting intact cells by chemical means include disruption by lytic enzymes, organic solvents and surfactants. Following mechanical or chemical disruption the cell fragments and other cellular debris are removed by centrifugation or membrane filtration.
Crude enzyme fractions or purified enzymes prepared from the disruption of intact cells can also be used in the method of the invention, satisfactorily, when the enzyme fractions or purified enzymes retain the essential activity, and include crude enzyme fractions or purified enzymes prepared from treated cell matter.
Exemplary microorganisms for use in the invention include those belonging to the following genera which may be isolated from natural origins, such as plant or soil material: Proteus, Providencia and Morganella. Particularly preferred microorganisms are those species within the genus Proteus, such as Proteus vulgaris ATCC 13315, Proteus mirabilis ATCC 29906, Proteus myxofaciens, ATCC 19692 and Proteus penneri ATCC 33519. Particularly preferred microorganisms are also those species within the genus Providencia, such as Providencia stuartii ATCC 33672. Particularly preferred microorganisms are those species within the genus Morganella, such as Morganella morganii ATCC 8019.
It should be understood that mutants of the biologically pure microorganisms are also contemplated by the present invention for use in the methods described herein, such as those modified by the use of chemical, physical (for example, x-rays) or biological means (for example, molecular biology techniques).
Growth of the microorganisms may be achieved by one of ordinary skill in the art without undue experimentation by the use of an appropriate medium, including solid and liquid media. Methods for culturing the microorganisms in accordance with the invention can be facilitated in conventionally used culture media, namely culture media containing a carbon source, nitrogen source, inorganic salts, trace metal salts, and vitamins. Furthermore, depending on the species or culture conditions of the microorganism, the ability to produce halo-L-tryptophan can be promoted by adding L-tryptophan at a concentration of about 1.0 to 10.0 g/l to the culture media. This range includes all specific values and sub-ranges there between, such as, but not limited to, 1.5, 2, 2.5, 3, 4, 5, 7 and 9 g/l. Furthermore, physiologically acceptable surfactants such as Triton X100 and olive oil may be added to the culture media, which sometimes serve to enhance the ability of the microorganisms to produce halo-L-tryptophan. As specific substances for use as the ingredients in the culture media, carbon sources such as glucose and sucrose, polyols such as glycerol, organic acids such as succinic acid, citric acid and fumaric acid, or mixtures thereof can be used. As the nitrogen source, ammonium sulfate, ammonium chloride, urea, yeast extract, meat extract, corn steep liquor and casein hydrolysate, or mixtures thereof can be used. As specific compositions of the culture media, for example, a culture medium containing 5 g/l succinic acid, 10 g/l casamino acid, 3 g/l yeast extract, 60 ml/l corn steep liquor, 5 g/l L-tryptophan, 5 g/l K2HPO4, 0.5 g/l MgSO4. 7H2O, 0.01 g/l FeSO4. 7H2O, 0.01 g/l MnSO4.4H2O, and 50 g/l Triton X100, pH 7.0.
The culture temperature to be used is within a range which the microorganism can generally grow, namely a range of 20 to 45xc2x0 C., preferably a range of 25 to 37xc2x0 C. This range includes all specific values and sub-ranges there between, such as, but not limited to, 21, 22, 23, 25, 27, 29, 30, 35, 37, 40 and 43xc2x0 C. Additionally, the culture media are adjusted within a range of pH 3 to 11, preferably within a range of pH 4 to 8. This range includes all specific values and sub-ranges there between, such as, but not limited to, pH 3.5, 4.5, 5, 5.5, 6, 6.5, 7, 9 and 10. Aeration conditions are set to aerobic or anaerobic conditions suitable for the growth of the microorganism to be used. The culture period is generally 12 to 120 hours, preferably about 24 to 96 hours. This range includes all specific values and sub-ranges there between, such as, but not limited to, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 and 110 hours.
Raw materials for the production of halo-L-tryptophan, namely a combination of haloindole and pyruvic acid and ammonia, or a combination of haloindole and a source supplying pyruvic acid and ammonia, such as L-serine, is collectively, intermittently or continuously added to the culture media. The raw materials can be added directly to the culture of the bacteria or can be added to the reaction mixture suspended with the cells or the treated cell matter separated from the culture. The substrates are added in the state of aqueous solution or slurry. To increase their solubility and to promote their dispersion, the substrates may be mixed with physiologically acceptable organic solvents and surfactants. Additionally, a remarkable outcome may sometimes be brought about when pyridoxal 5xe2x80x2-phosphate is added at a concentration of from 0.01 mg/ml to 0.3 mg/ml. This range includes all specific values and sub-ranges there between, such as, but not limited to, 0.02, 0.03, 0.05, 0.075, 0.1, 0.15, 0.2, 0.25, 0.275 mg/ml.
To produce halo-L-tryptophan using the culture method, raw materials for producing halo-L-tryptophan are added to the culture, as they are, so as to continue the culturing. Any amount of haloindole can be added, with no limitation. However, the amount is generally at a concentration of 1 g/l to 200 g/l. This range includes all specific values and sub-ranges there between, such as, but not limited to, 2, 5, 10, 20, 40, 50, 100, 150 and 175 g/l. In this case, the pH of the culture medium after addition of the raw materials is adjusted to about pH 8.4 to 9.0, which works to enhance the productivity of halo-L-tryptophan.
To produce halo-L-tryptophan using cells isolated from culture the bacteria are first separated from the culture medium and then suspended or dissolved together with the raw materials including haloindole in aqueous media, to produce halo-L-tryptophan. Haloindole can be used at any amount with no specific limitation, which is generally adjusted to a concentration of 1 g/l to 200 g/l. This range includes all specific values and sub-ranges there between, such as, but not limited to, 2, 5, 10, 20, 40, 50, 100, 150 and 175 g/l. The reaction is carried out at a temperature of 20 to 60xc2x0 C., preferably within a range of 30 to 45xc2x0 C. This range includes all specific values and sub-ranges there between, such as, but not limited to, 25, 30, 35, 40, 45, 50 and 55xc2x0 C. Additionally, the reaction solution is adjusted to a range of pH 7 to 11, preferably pH 8 to 9.5. This range includes all specific values and sub-ranges there between, such as, but not limited to, pH 7.3, 7.5, 7.8, 8.0, 8.5, 9.0, 9.5, 10 and 10.5. The reaction time is generally one to 120 hours, preferably about 6 to 96 hours. This range includes all specific values and sub-ranges there between, such as, but not limited to, 2, 5, 10, 20, 30, 40, 50, 75, 100 and 110 hours.
The resulting halo-L-tryptophan can be determined quantitatively by well-known methods in a rapid manner. More specifically, high performance liquid chromatography on an ODS column is satisfactorily used. For the determination of optical purity, high performance liquid chromatography using an optical resolution column such as CROWNPAK CR (+) manufactured by Daicel Chemical Industry may satisfactorily be used. The halo-L-tryptophan which accumulates in the culture medium or the reaction solution can be collected from the culture or the reaction solution by conventional methods. The halo-L-tryptophan can be collected from the culture or the reaction solution by methods conventionally known. Procedures such as filtration, centrifugation, concentration in vacuum, solvent extraction, ion exchange or adsorption chromatography and crystallization are appropriately combined together and used, depending on the need.